9-aminonoscapine and its use in treating cancers, including drug-resistant cancers

ABSTRACT

9-aminonoscapine, prodrugs thereof, and pharmaceutically acceptable salts thereof, are disclosed. Pharmaceutical compositions including 9-aminonoscapine, and methods of preparation and use thereof are disclosed. 9-aminonoscapine is a noscapine analog that can be used to treat and/or prevent a wide variety of cancers, including drug resistant cancers, by binding tubulin and inducing apoptosis selectively in tumor cells (ovarian and T-cell lymphoma) resistant to paclitaxel, vinblastine and teniposide. 9-aminonoscapine can perturb the progression of cell cycle by mitotic arrest, followed by apoptotic cell death associated with increased caspase-3 activation and appearance of TUNEL-positive cells. Thus, 9-aminonoscapine is a novel therapeutic agents for a variety of cancers, including ovarian and T-cell lymphoma cancers, even those that have become drug-resistant to currently available chemotherapeutic drugs.

The U.S. government has certain rights to this invention pursuant to NIHgrant No. 1 R01 CA095317-01A2.

FIELD OF THE INVENTION

The present invention relates to the noscapine analog 9-aminonoscapine,pharmaceutical compositions incorporating 9-aminonoscapine analog, andmethods of using the compound and compositions to treat cancers,including drug resistant cancers.

BACKGROUND OF THE INVENTION

Microtubules are major cytoskeletal structures responsible formaintaining genetic stability during cell division (Sammak and Borisy,1987; McIntosh, 1994;

Desai and Mitchinson, 1997). The dynamics of these polymers isabsolutely crucial for this function that can be described as theirgrowth rate at the plus ends, catastrophic shortening, frequency oftransition between the two phases, pause between the two phases, theirrelease from the microtubule organizing center and treadmilling(Margolis and Wilson, 1981; Mitchison and Kirschner, 1984; Kirschner andMitchison, 1986; Margolis and Wilson, 1998; Jordan and Wilson, 2004).Microtubule lattice also serves as tracks for the axonal transport oforganelles driven by anteriograde and retrograde molecular motors togenerate and maintain axonal integrity (Joshi, 1998; Nogales, 2000).Interference with microtubule dynamics often leads to programmed celldeath and thus microtubule-binding drugs are currently used to treatvarious malignancies in the clinic (Jordan and Wilson, 2004). Althoughuseful, currently used microtubule drugs such as vincas and taxanes arelimited due to the emergence of drug resistance. There have beenmultiple mechanisms for antimicrotubule drug resistance includingoverexpression of drug-efflux pumps, misexpression of tubulin isotypes,and perhaps mutational lesions in tubulin itself (Ranganathan et al.,1996; Giannakakou et al., 1997; Monzo et al., 1999; Dumontet et al.,2005).

The pharmacological profile of microtubule-binding agents, however, hasnot been ideal. Most of them need to be infused over long periods oftime in the clinic because they are not water-soluble, and can causehypersensitive reactions due to the vehicle solution (Rowinsky, 1997).Furthermore, normally dividing cells within the healthy tissues such asintestinal crypts, hair follicles, and the bone marrow are alsovulnerable to these agents, leading to toxicities (Rowinsky, 1997). Inaddition, nerve cells dependent on molecular traffic over long distancesundergo degenerative changes causing peripheral neuropathies (Pace etal., 1996; Crown and O'Leary, 2000; Theiss and Meller, 2000; Topp etal., 2000).

Noscapine((S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-5,6,7,8-tetrahydro[1,3]-dioxolo-[4,5-g]isoquinolin-5-yl)isobenzo-furan-1(3H)-one),a safe antitussive agent for over 40 years, binds tubulin, arrestsdividing cells in mitosis and induces apoptosis (Ye et al., 1998). It iswell-tolerated in humans and has been shown to be non-toxic in healthyvolunteers, including pregnant mothers (Dahlstrom et al., 1982; Karlssonet al., 1990; Jensen et al., 1992).

Unlike the other microtubule-targeting drugs, noscapine does notsignificantly change the microtubule polymer mass even at highconcentrations. Instead, it suppresses microtubule dynamics byincreasing the time that microtubules spend in an attenuated (pause)state when neither microtubule growth nor shortening is detectable(Landen et al., 2002). Thus, noscapine-induced suppression ofmicrotubule dynamics, even though subtle, is sufficient to interferewith the proper attachment of chromosomes to kinetochore microtubulesand to suppress the tension across paired kinetochores (Zhou et al.,2002a). This represents an improvement over the taxanes, themicrotubule-bundling agents or overpolymerizers, and vincas, thedepolymerizers, that cause toxicities in mitotic and post mitoticneurons at elevated doses.

Noscapine thus effectively inhibits the progression of various cancertypes both in cultured cells and in animal models with no obvious sideeffects (Ye et al., 1998; Landen et al., 2002; Zhou et al., 2002b; 2003;Landen et al., 2004). Surprisingly, the apoptosis is much morepronounced in cancer cells compared with normal healthy cells (Landen etal., 2002).

It would be desirable to have compounds, compositions and methods forpreventing and/or treating various types of cancer, without significantassociated side effects, that provide increased anti-cancer propertiesto that of noscapine. The present invention provides such a compound,compositions and methods.

SUMMARY OF THE INVENTION

9-aminonoscapine, pharmaceutically acceptable salts, prodrugs andmetabolites thereof, (herein referred to as the “compounds”),pharmaceutical compositions including the compounds, and methods ofpreparation and use thereof are disclosed. 9-aminonoscapine is anoscapine analog, with an amine group at the 9-position of theisoquinoline ring. The synthesis, characterization and an evaluation ofthe anti-tumor potential of 9-amino-Nos is described herein.

9-aminonoscapine binds tubulin, and effectively inhibits cellproliferation of 1A9 (ovarian cancer cells) and its paclitaxel-resistantvariant (1A9/PTX22), and human lymphoblastoid cells CEM, and itsvinblastine-(CEM/VLB100) and teniposide-(CEM/VM-1-5) resistant variants.

Treatment with 9-aminonoscapine halts cell cycle progression in cellsdue to the checkpoints governed by many crucial genes that are mutatedin cancer cells. Therefore, we hypothesize that normal cells resumenormal cell cycle as the drug clears from the system (excretion,metabolism etc). However, due to checkpoint lesions in cancer cells,they do not arrest for longer times but undergo mitotic catastrophic andapoptosis. Therefore, 9-aminonoscapine only affects cancer cells andspares the normal cells.

The pharmaceutical compositions include an effective amount of thecompounds described herein, along with a pharmaceutically acceptablecarrier or excipient. When employed in effective amounts, the compoundscan act as a therapeutic agent to prevent and/or treat a wide variety ofcancers, particularly drug resistant cancers, and are believed to beboth safe and effective in this role. Representative cancers that can betreated and/or prevented include drug-resistant ovarian cancer, drugresistant T-cell lymphoma, leukemia, non-small cell lung, colon, centralnervous system (CNS), melanoma, renal, ovarian, breast and prostatecancer.

The foregoing and other aspects of the present invention are explainedin detail in the detailed description and examples set forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D are charts showing the fluorescence quenching of tubulin by9-aminonoscapine. In FIG. 1A, the tubulin fluorescence emission spectrumis quenched by Nos, and in FIG. 1B, the tubulin fluorescence emissionspectrum is quenched by 9-aminonoscapine, in a concentration-dependentmanner. FIG. 1C is a double reciprocal plot showing a dissociationconstant (K_(d)) of 152±1 μM for Nos binding to tubulin, and FIG. 1D isa double reciprocal plot showing a dissociation constant (K_(d)) of 14±1μM for 9-aminonoscapine binding to tubulin. Values are mean±SD for fourexperiments performed in triplicate (p<0.05). The graphs shown are arepresentative of four experiments performed.

FIG. 2 is a bar graph showing the IC₅₀ values for the in vitro treatmentof various cancer cell types with 9-aminonoscapine (μM)

DETAILED DESCRIPTION OF THE INVENTION

Compounds, pharmaceutical compositions including the compounds, andmethods of preparation and use thereof are disclosed.

The following definitions will be useful in understanding the metes andbounds of the invention as described herein.

I. 9-Aminonoscapine

The compounds described herein include 9-aminonoscapine((S)-6,7-dimethoxy-3-((R)-4-methoxy-6-methyl-9-amino-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)isobenzofuran-1(3H)-one)),a noscapine analog with an amine group at the 9-position of thequinoline ring, prodrugs or metabolites of this compound, andpharmaceutically acceptable salts thereof. 9-amino-noscapine has thestructure shown below.

The compound can exist in varying degrees of enantiomeric excess.

The compound can be in a free base form or in a salt form (e.g., aspharmaceutically acceptable salts).

Pharmaceutically-Acceptable Salts

Examples of suitable pharmaceutically acceptable salts include inorganicacid addition salts such as sulfate, phosphate, and nitrate; organicacid addition salts such as acetate, galactarate, propionate, succinate,lactate, glycolate, malate, tartrate, citrate, maleate, fumarate,methanesulfonate, p-toluenesulfonate, and ascorbate; salts with anacidic amino acid such as aspartate and glutamate; alkali metal saltssuch as sodium and potassium; alkaline earth metal salts such asmagnesium and calcium; ammonium salt; organic basic salts such astrimethylamine, triethylamine, pyridine, picoline, dicyclohexylamine,and N,N′-dibenzylethylenediamine; and salts with a basic amino acid suchas lysine and arginine. The salts can be in some cases hydrates orethanol solvates. The stoichiometry of the salt will vary with thenature of the components.

Pharmaceutically acceptable salts may be obtained using standardprocedures well known in the art, for example by reacting the aminegroup with a suitable acid affording a physiologically acceptable anion.In one embodiment, the salt is a hydrochloride salt of the compound.

Prodrugs and Derivatives

The active compound can be administered as any salt or prodrug that uponadministration to the recipient is capable of providing directly orindirectly the parent compound, or that exhibits activity itself.Non-limiting examples include forms of 9-aminonoscapine in which theamine group has been alkylated, acylated, or otherwise modified (a typeof “pharmaceutically acceptable prodrug”).

Further, the modifications can affect the biological activity of thecompound, in some cases increasing the activity over the parentcompound. This can easily be assessed by preparing the salt or prodrugand testing its anticancer or other activity according to the methodsdescribed herein, or other methods known to those skilled in the art.

Prodrug forms of the compound include the following types of derivativeswhere each R group individually can be hydrogen, substituted orunsubstituted alkyl, aryl, alkenyl, alkynyl, heterocycle, alkylaryl,aralkyl, aralkenyl, aralkynl, cycloalkyl or cycloalkenyl groups.

(a) Carboxamides, —NHC(O)R

(b) Carbamates, —NHC(O)OR

(c) (Acyloxy)alkyl Carbamates, NHC(O)OROC(O)R

(d) Enamines, —NHCR(═CHCO₂R) or —NHCR(═CHCONR₂)

(e) Schiff Bases, —N═CR₂

(f) Mannich Bases (from carboximide compounds), RCONHCH₂NR₂

As used herein, alkyl refers to C₁₋₈ straight, branched, or cyclic alkylgroups, and alkenyl and alkynyl refers to C₂₋₈ straight, branched orcyclic moieties that include a double or triple bond, respectively. Arylgroups include C₆₋₁₀ aryl moieties, specifically including benzene.Heterocyclic groups include C₃₋₁₀ rings which include one or more O, N,or S atoms. Alkylaryl groups are alkyl groups with an aryl moiety, andthe linkage to the nitrogen at the 9-position on the noscapine frameworkis through a position on the alkyl group. Arylalkyl groups are arylgroups with an alkyl moiety, and the linkage to the nitrogen at the9-position on the noscapine framework is through a position on the arylgroup. Aralkenyl and aralkynyl groups are similar to aralkyl groups,except that instead of an alkyl moiety, these include an alkenyl oralkynyl moiety. Substituents for each of these moieties include halo,nitro, amine, thio, hydroxy, ester, thioester, ether, aryl, alkyl,carboxy, amide, azo, sulfonyl, and

Other prodrugs include prodrugs that are converted in biological milieuvia ester hydrolysis via an enzymatic route rather than chemicalhydrolysis, for example, by serine-dependent esterases. Representativeprodrugs of this type are described, for example, in Amsberry et al.,“Amine Prodrugs Which Utilize Hydroxy Amide Lactonization. II. APotential Esterase-Sensitive Amide Prodrug,” Pharmaceutical Research,Volume 8(4): 455-461(7) (April 1991).

Azo-based prodrugs can also be used. For example, bacterial reductasescan use reductive cleavage to convert the following azo prodrug in vivoto the active form.

II. Method of 9-Aminonoscapine Synthesis

Experimental

General:

¹H NMR and ¹³C NMR spectra were measured in CDCl₃ on INOVA 400 NMRspectrometer. All proton NMR spectra were recorded at 400 MHz and werereferenced with residual chloroform (7.27 ppm). All carbon NMR spectrawere recorded at 100 MHz and were referenced with 77.27 ppm resonance ofresidual chloroform. Abbreviations for signal coupling are as follows:s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Infraredspectra were recorded on sodium chloride discs on Mattson Genesis IIFT-IR. High resolution mass spectra were collected on Thermo FinniganLTQ-FT Hybrid mass spectrophotometer using 3-nitrobenzyl alcohol, insome cases with addition of LiI as a matrix. Melting points weredetermined using a Thomas Hoover melting point apparatus and wereuncorrected. All reactions were conducted in oven-dried (125° C.)glassware under an atmosphere of dry argon. All common reagents andsolvents were obtained from commercial suppliers and used withoutfurther purification unless otherwise indicated. Solvents were dried bystandard methods. The reactions were monitored by thin layerchromatography (TLC) using silica gel 60 F254 (Merck) precoated aluminumsheets. Flash chromatography was carried out on standard grade silicagel (230-400 mesh).

Synthesis of 9-aminonoscapine was shown in Scheme 1. Briefly, noscapine(1) was dissolved minimum amount of 48% hydrobromic acid and thencautiously added freshly prepared bromine water. The reaction mixturestirred for 1 h at 25° C. and the resultant mixture was basified to pH10 to afford 9-bromonoscapine in 82% yield. Refluxing compound 2 in DMFwith sodium azide and sodium iodide for 15 hours gave its azidoderivative (3) in quantitative yield. Reduction of azido derivative withtin chloride in the presence of thiophenol and triethylamine in THF for2h at 25° C. afforded the title compound, 9-aminonoscapine (4) in 83%yield.

(S)-3-((R)-9-bromo-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one(2): To a flask containing noscapine (20 g, 48.4 mmol) was added minimumamount of 48% hydrobromic acid solution (˜40 ml) to dissolve or make asuspension of the reactant. To the reaction mixture was added freshlyprepared bromine water (˜250 ml) drop wise until an orange precipitateappeared. The reaction mixture was then stirred at room temperature for1 h to attain completion, neutralized to pH 10 using ammonia solution toafford solid precipitate. The solid precipitate was recrystallized withethanol to afford bromo-substituted noscapine. Yield: 82%; mp 169-170°C.; IR: 2945 (m), 2800 (m), 1759 (s), 1612 (m), 1500 (s), 1443 (s), 1263(s), 1091 (s), 933 (w) cm⁻¹; ¹H NMR (CDCl₃, 400 MHz), δ 7.04 (d, 1H, J=7Hz), 6.32 (d, 1H, J=7 Hz), 6.03 (s, 2H), 5.51(d, 1H, J=4 Hz), 4.55 (d,1H, J=4 Hz), 4.10 (s, 3H), 3.98 (s, 3H), 3.89 (s, 3H), 2.52 (s, 3H),2.8-1.93 (m, 4H); ¹³C NMR (CDCl₃, 100 MHz), δ 167.5, 151.2, 150.5,150.1, 148.3, 140.0, 135.8, 130.8, 120.3, 120.4, 120.1, 105.3, 100.9,100.1, 87.8, 64.4, 56.1, 56.0, 55.8, 51.7, 41.2, 27.8; MS (FAB): m/z(relative abundance, %), 494 (93.8), 492 (100), 300 (30.5), 298 (35.4);MALDI: m/z 491.37 (M+), 493.34; ESI/tandem mass spectrometry: parent ionmasses, 494, 492; daughter ion masses (intensity, %), 433 (51), 431(37), 300 (100), 298 (93.3); HRMS (ESI): m/z Calcd. for C₂₂H₂₃BrNO₇(M+1), 493.3211; Found, 493.3215 (M+1).

(S)-3-((R)-9-azido-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one(3):

To a solution of compound 2 (2.0 g, 4.063 mmol) in DMF (20 mL) wereadded sodium azide (2.641 g, 40.63 mmol) and sodium iodide (0.609 g,4.063 mmol) and the reaction mixture was stirred at 80° C. for 15 h toattain completion. Then the solvent was removed in vacuo and theresultant residue was dissolved in chlorofrom (40 mL), washed with water(2×40 mL), dried over sodium sulfate and concentrated to obtain thetitled compound 3, which was recrystallized with ethanol in hexane(10:90) to afford brown crystals. Yield, 89%; mp 177.2-178.1° C.; IR:1529, 1362 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz): δ 7.05 (d, 1H, J=7.0 Hz), 6.4(d, 1H, J=7.0 Hz), 6.01 (s, 2H), 5.85 (d, 1H, J=4.4 Hz), 4.40 (d, 1H,J=4.4 Hz), 4.15 (s, 3H), 3.88 (s, 3H), 3.84 (s, 3H), 2.75-2.62 (m, 2H),2.60-2.56 (m, 2H), 2.51 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.2,157.7, 152.6, 147.9, 142.2, 140.5, 135.0, 134.0, 123.5, 121.8, 119.7,119.3, 114.1, 100.5, 87.4, 64.1, 56.7, 56.5, 56.2, 51.4, 39.2, 27.2;HRMS (ESI): m/z Calcd. for C₂₂H₂₃N₄O₇ (M+1), 455.4335; Found, 455.4452(M+1).

(S)-3-((R)-9-amino-4-methoxy-6-methyl-5,6,7,8-tetrahydro-[1,3]dioxolo[4,5-g]isoquinolin-5-yl)-6,7-dimethoxyisobenzofuran-1(3H)-one(4): To a 50-mL of round-bottomed flask containing a solution of SnCl₂in THF (10 mL) were added thiophenol and triphenylamine. The reactionmixture was added slowly to a solution of azido-noscapine (3, 0.2 g,0.440 mmol) in THF (5 mL) and the reaction mixture stirred at roomtemperature. The reaction progress was monitored by thin-layerchromatography at 30 minutes intervals. The reaction was found to becompleted after 2 h, the solvent was removed in vacuo. The residue wasdiluted with chloroform (20 ml) and was added sodium hydroxidesolution(20 mL). the aqueous phase was separated and extracted withchloroform (2×20 mL). the combined organic phase was dried over sodiumsulfate and concentrated to obtain amino-noscapine as colorless oil,which was then treated with ethereal HCl to obtain its salt as whitecrystals. Yield, 83%; mp (HCl. Salt) 112.2-112.6° C.; IR: 1725, 1362cm⁻¹; ¹H NMR (CDCl₃, 400 MHz): δ 7.12 (d, 1H, J=7.4.0 Hz), 7.02 (d, 1H,J=7.4 Hz), 6.02 (s, 2H), 5.92 (d, 1H, J=4.0 Hz), 4.42 (d, 1H, J=4.0 Hz),4.20 (bs, 2H), 4.02 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 2.74-2.64 (m,2H), 2.61-2.56 (m, 2H), 2.52 (s, 3H); ¹H NMR (CDCl₃+D₂O, 400 MHz): δ7.12 (d, 1H, J=7.4.0 Hz), 7.02 (d, 1H, J=7.4 Hz), 6.02 (s, 2H), 5.92 (d,1H, J=4.0 Hz), 4.42 (d, 1H, J=4.0 Hz), 5.12 (bs, confirms NH₂ group),4.02 (s, 3H), 3.85 (s, 3H), 3.80 (s, 3H), 2.74-2.64 (m, 2H), 2.61-2.56(m, 2H), 2.52 (s, 3H); ¹³C NMR (CDCl₃, 100 MHz): δ 169.5, 156.8, 152.6,147.8, 142.7, 141.8, 135.0, 134.2, 123.2, 120.8, 119.9, 119.4, 114.1,100.8, 87.6, 63.7, 56.8, 56.4, 56.1, 51.4, 39.2, 27.5; HRMS (ESI): m/zCalcd. for C₂₂H₂₄N₂O₇ (M+1), 428.3481; Found, 428.1562 (M+1).

HPLC Purity and Peak Attributions:

The HPLC purity was determined following two different methods usingvaried solvent systems.

Method 1: Ultimate Plus, LC Packings, Dionex, C₁₈ column (pep Map 100, 3μm, 100 Å particle size, ID: 1000 μm, length: 15 cm) with solventsystems A (0.1% formic acid in water) and B (acetonitrile), gradient, 25min run at a flow of 40 μL/min. Retention time for 9-amino-nos is 18.30min. HPLC purity was 95%.

Method 2: Ultimate Plus, LC Packings, Dionex, C₁₈ column (pep Map 100, 3μm, 100 Å particle size, ID: 1000 μm, length: 15 cm) with solventsystems A (0.1% formic acid in water) and B (methanol), gradient, 25 minrun at a flow of 40 μL/min. Retention time for 9-amino-nos is 18.96 min.HPLC purity was 94%.

Other possible synthetic methods involve nitrating the aromatic ring,and reducing the nitrate group to an amine group. Such nitration andreduction reactions are well known to those of skill in the art.Ideally, methods do not involve reagents which reduce or hydrolyze thelactone moiety. In some embodiments, the lactone can be protected with asuitable protecting group, the nitro group reduced to an amine, and thelactone deprotected.

In other embodiments, the nitro group can be converted to a diazoniumsalt, followed by displacement to form the amine.

Other amines than 9-NH₂ can be formed, for example, by first forming the9-noscapine, and then converting the 9-NH₂ group to another moiety usingalkylation reagents in alkylation reactions. Suitable alkylationreagents as are known in the art, and include C₁₋₈ alkyl halides, suchas alkyl bromides and iodides.

Conclusions:

Relatively simple and straightforward methods for the direct, andregioselective nitration of noscapine, which provide the nitratedproduct in high quantitative yields, are provided herein. A plethora ofreagents and reaction conditions have been reported for reduction ofaromatic nitro groups to form aromatic amine groups, though appropriateconditions need to be selected for the noscapine framework, as it isreadily hydrolysable. These synthetic strategies effect the desiredtransformations under mild conditions.

III. Pharmaceutical Compositions

The compound, 9-aminonoscapine, and its prodrugs and metabolites, andpharmaceutically acceptable salts, as described herein, can beincorporated into pharmaceutical compositions and used to treat orprevent a condition or disorder in a subject susceptible to such acondition or disorder, and/or to treat a subject suffering from thecondition or disorder. Optically active compounds can be employed asracemic mixtures, as pure enantiomers, or as compounds of varyingenantiomeric purity. The pharmaceutical compositions described hereininclude 9-aminonoscapine, and its prodrugs and metabolites, andpharmaceutically acceptable salts, as described herein, and apharmaceutically acceptable carrier and/or excipient.

The manner in which the compounds are administered can vary. Thecompositions are preferably administered orally (e.g., in liquid formwithin a solvent such as an aqueous or non-aqueous liquid, or within asolid carrier). Preferred compositions for oral administration includepills, tablets, capsules, caplets, syrups, and solutions, including hardgelatin capsules and time-release capsules. Compositions may beformulated in unit dose form, or in multiple or subunit doses. Preferredcompositions are in liquid or semisolid form. Compositions including aliquid pharmaceutically inert carrier such as water or otherpharmaceutically compatible liquids or semisolids may be used. The useof such liquids and semisolids is well known to those of skill in theart.

The compositions can also be administered via injection, i.e.,intraveneously, intramuscularly, subcutaneously, intraperitoneally,intraarterially, intrathecally; and intracerebroventricularly.Intravenous administration is a preferred method of injection. Suitablecarriers for injection are well known to those of skill in the art, andinclude 5% dextrose solutions, saline, and phosphate buffered saline.The compounds can also be administered as an infusion or injection(e.g., as a suspension or as an emulsion in a pharmaceuticallyacceptable liquid or mixture of liquids).

The formulations may also be administered using other means, forexample, rectal administration. Formulations useful for rectaladministration, such as suppositories, are well known to those of skillin the art. The compounds can also be administered by inhalation (e.g.,in the form of an aerosol either nasally or using delivery articles ofthe type set forth in U.S. Pat. No. 4,922,901 to Brooks et al., thedisclosure of which is incorporated herein in its entirety); topically(e.g., in lotion form); or transdermally (e.g., using a transdermalpatch, using technology that is commercially available from Novartis andAlza Corporation). Although it is possible to administer the compoundsin the form of a bulk active chemical, it is preferred to present eachcompound in the form of a pharmaceutical composition or formulation forefficient and effective administration.

Exemplary methods for administering such compounds will be apparent tothe skilled artisan. The usefulness of these formulations may depend onthe particular composition used and the particular subject receiving thetreatment. These formulations may contain a liquid carrier that may beoily, aqueous, emulsified or contain certain solvents suitable to themode of administration.

The compositions can be administered intermittently or at a gradual,continuous, constant or controlled rate to a warm-blooded animal (e.g.,a mammal such as a mouse, rat, cat, rabbit, dog, pig, cow, or monkey),but advantageously are administered to a human being. In addition, thetime of day and the number of times per day that the pharmaceuticalformulation is administered can vary.

Preferably, the compositions are administered such that activeingredients interact with regions where cancer cells are located. Thecompounds described herein are very potent at treating these cancers.

In certain circumstances, the compounds described herein can be employedas part of a pharmaceutical composition with other compounds intended toprevent or treat a particular cancer, i.e., combination therapy. Inaddition to effective amounts of the compounds described herein, thepharmaceutical compositions can also include various other components asadditives or adjuncts.

Combination Therapy

The combination therapy may be administered as (a) a singlepharmaceutical composition which comprises 9-aminonoscapine as describedherein, or its prodrugs or metabolites, or pharmaceutically acceptablesalts, at least one additional pharmaceutical agent described herein,and a pharmaceutically acceptable excipient, diluent, or carrier; or (b)two separate pharmaceutical compositions comprising (i) a firstcomposition comprising 9-aminonoscapine as described herein and apharmaceutically acceptable excipient, diluent, or carrier, and (ii) asecond composition comprising at least one additional pharmaceuticalagent described herein and a pharmaceutically acceptable excipient,diluent, or carrier. The pharmaceutical compositions can be administeredsimultaneously or sequentially and in any order.

In use in treating or preventing cancer, 9-aminonoscapine can beadministered together with at least one other chemotherapeutic agent aspart of a unitary pharmaceutical composition. Alternatively, it can beadministered apart from the other anticancer chemotherapeutic agent. Inthis embodiment, 9-aminonoscapine and the at least one other anticancerchemotherapeutic agent are administered substantially simultaneously,i.e. the compounds are administered at the same time or one after theother, so long as the compounds reach therapeutic levels for a period oftime in the blood.

Combination therapy involves administering 9-aminonoscapine, asdescribed herein, or a pharmaceutically acceptable salt or prodrug of9-aminonoscapine, in combination with at least one anti-cancerchemotherapeutic agent, ideally one which functions by a differentmechanism (i.e., VEGF inhibitors, alkylating agents, and the like).

Examples of known anticancer agents which can be used for combinationtherapy include, but are not limited to alkylating agents, such asbusulfan, cis-platin, mitomycin C, and carboplatin; antimitotic agents,such as colchicine, vinblastine, paclitaxel, and docetaxel; topo Iinhibitors, such as camptothecin and topotecan; topo II inhibitors, suchas doxorubicin and etoposide; RNA/DNA antimetabolites, such as5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites,such as 5-fluoro-2′-deoxy-uridine, ara-C, hydroxyurea and thioguanine;and antibodies, such as Herceptin® and Rituxan®. Other known anti-canceragents, which can be used for combination therapy, include arsenictrioxide, gamcitabine, melphalan, chlorambucil, cyclophosamide,ifosfamide, vincristine, mitoguazone, epirubicin, aclarubicin,bleomycin, mitoxantrone, elliptinium, fludarabine, octreotide, retinoicacid, tamoxifen and alanosine. Other classes of anti-cancer compoundsthat can be used in combination with 9-aminonoscapine are describedbelow.

9-aminonoscapine can be combined with alpha-1-adrenoceptor antagonists,such as doxazosin, terazosin, and tamsulosin, which can inhibit thegrowth of prostate cancer cell via induction of apoptosis (Kyprianou,N., et al., Cancer Res 60:4550 4555, (2000)).

Sigma-2 receptors are expressed in high densities in a variety of tumorcell types (Vilner, B. J., et al., Cancer Res. 55: 408 413 (1995)) andsigma-2 receptor agonists, such as CB-64D, CB-184 and haloperidol,activate a novel apoptotic pathway and potentiate antineoplastic drugsin breast tumor cell lines. (Kyprianou, N., et al., Cancer Res. 62:313322 (2002)). Accordingly, 9-aminonoscapine can be combined with at leastone known sigma-2 receptor agonists, or a pharmaceutically acceptablesalt of said agent.

9-aminonoscapine can be combined with lovastatin, a HMG-CoA reductaseinhibitor, and butyrate, an inducer of apoptosis in the Lewis lungcarcinoma model in mice, can potentiate antitumor effects (Giermasz, A.,et al., Int. J. Cancer 97:746 750 (2002)). Examples of known HMG-CoAreductase inhibitors, which can be used for combination therapy include,but are not limited to, lovastatin, simvastatin, pravastatin,fluvastatin, atorvastatin and cerivastatin, and pharmaceuticallyacceptable salts thereof.

Certain HIV protease inhibitors, such as indinavir or saquinavir, havepotent anti-angiogenic activities and promote regression of Kaposisarcoma (Sgadari, C., et al., Nat. Med. 8:225 232 (2002)). Accordingly,9-aminonoscapine can be combined with HIV protease inhibitors, or apharmaceutically acceptable salt of said agent. Representative HIVprotease inhibitors include, but are not limited to, amprenavir,abacavir, CGP-73547, CGP-61755, DMP-450, indinavir, nelfinavir,tipranavir, ritonavir, saquinavir, ABT-378, AG 1776, and BMS-232,632.

Synthetic retinoids, such as fenretinide (N-(4-hydroxyphenyl)retinamide,4HPR), can have good activity in combination with other chemotherapeuticagents, such as cisplatin, etoposide or paclitaxel in small-cell lungcancer cell lines (Kalemkerian, G. P., et al., Cancer Chemother.Pharmacol. 43:145 150 (1999)). 4HPR also was reported to have goodactivity in combination with gamma-radiation on bladder cancer celllines (Zou, C., et al., Int. J. Oncol. 13:1037 1041 (1998)).Representative retinoids and synthetic retinoids include, but are notlimited to, bexarotene, tretinoin, 13-cis-retinoic acid, 9-cis-retinoicacid, .alpha.-difluoromethylornithine, ILX23-7553, fenretinide, andN-4-carboxyphenyl retinamide.

Proteasome inhibitors, such as lactacystin, exert anti-tumor activity invivo and in tumor cells in vitro, including those resistant toconventional chemotherapeutic agents. By inhibiting NF-kappaBtranscriptional activity, proteasome inhibitors may also preventangiogenesis and metastasis in vivo and further increase the sensitivityof cancer cells to apoptosis (Almond, J. B., et al., Leukemia 16:433 443(2002)). Representative proteasome inhibitors include, but are notlimited to, lactacystin, MG-132, and PS-341.

Tyrosine kinase inhibitors, such as STI571 (Imatinib mesilate,Gleevec®), have potent synergetic effects in combination with otheranti-leukemic agents, such as etoposide (Liu, W. M., et al. Br. J.Cancer 86:1472 1478 (2002)). Representative tyrosine kinase inhibitorsinclude, but are not limited to, Gleevec®, ZD1839 (Iressa®), SH268,genistein, CEP2563, SU6668, SU11248, and EMD121974.

Prenyl-protein transferase inhibitors, such as farnesyl proteintransferase inhibitor R115777, possess antitumor activity against humanbreast cancer (Kelland, L. R., et. al., Clin. Cancer Res. 7:3544 3550(2001)). Synergy of the protein farnesyltransferase inhibitor SCH66336and cisplatin in human cancer cell lines also has been reported (Adjei,A. A., et al., Clin. Cancer. Res. 7:1438 1445 (2001)). Prenyl-proteintransferase inhibitors, including farnesyl protein transferaseinhibitor, inhibitors of geranylgeranyl-protein transferase type I(GGPTase-I) and geranylgeranyl-protein transferase type-II, or apharmaceutically acceptable salt of said agent, can be used incombination with 9-aminonoscapine. Examples of known prenylproteintransferase inhibitors include, but are not limited to, R115777,SCH66336, L-778,123, BAL9611 and TAN-1813.

Cyclin-dependent kinase (CDK) inhibitors, such as flavopiridol, havepotent, often synergetic, effects in combination with other anticanceragents, such as CPT-11, a DNA topoisomerase I inhibitor in human coloncancer cells (Motwani, M., et al., Clin. Cancer Res. 7:4209 4219,(2001)). Representative cyclin-dependent kinase inhibitors include, butare not limited to, flavopiridol, UCN-01, roscovitine and olomoucine.

Certain COX-2 inhibitors are known to block angiogenesis, suppress solidtumor metastases, and slow the growth of implanted gastrointestinalcancer cells (Blanke, C. D., Oncology (Huntingt) 16(No. 4 Suppl. 3):1721 (2002)). Representative COX-2 inhibitors include, but are not limitedto, celecoxib, valecoxib, and rofecoxib.

Any of the above-mentioned compounds can be used in combination therapywith the noscapine analogues. Further, 9-aminonoscapine can be targetedto a tumor site by conjugation with therapeutically useful antibodies,such as Herceptin® or Rituxan®, growth factors, such as DGF, NGF;cytokines, such as IL-2, IL-4, or any molecule that binds to the cellsurface. The antibodies and other molecules will deliver a compounddescribed herein to its targets and make it an effective anticanceragent. The bioconjugates can also enhance the anticancer effect oftherapeutically useful antibodies, such as Herceptin® or Rituxan®.

The compounds can also be used in conjunction with surgical tumorremoval, by administering the compounds before and/or after surgery, andin conjunction with radiation therapy, by administering the compoundsbefore, during, and/or after radiation therapy.

The appropriate dose of the compound is that amount effective to preventoccurrence of the symptoms of the disorder or to treat some symptoms ofthe disorder from which the patient suffers. By “effective amount”,“therapeutic amount” or “effective dose” is meant that amount sufficientto elicit the desired pharmacological or therapeutic effects, thusresulting in effective prevention or treatment of the disorder.

When treating cancers, an effective amount of the noscapine analogue isan amount sufficient to suppress the growth of the tumor(s), and,ideally, is a sufficient amount to shrink the tumor, and, more ideally,to destroy the tumor. Cancer can be prevented, either initially, or fromre-occurring, by administering the compounds described herein in aprophylactic manner. Preferably, the effective amount is sufficient toobtain the desired result, but insufficient to cause appreciable sideeffects.

The effective dose can vary, depending upon factors such as thecondition of the patient, the severity of the cancer, and the manner inwhich the pharmaceutical composition is administered. The effective doseof compounds will of course differ from patient to patient, but ingeneral includes amounts starting where desired therapeutic effectsoccur but below the amount where significant side effects are observed.

The compounds, when employed in effective amounts in accordance with themethod described herein, are selective to certain cancer cells, but donot significantly affect normal cells.

For human patients, the effective dose of typical compounds generallyrequires administering the compound in an amount of at least about 1,often at least about 10, and frequently at least about 25 μg/24hr/patient. The effective dose generally does not exceed about 500,often does not exceed about 400, and frequently does not exceed about300 μg/24 hr/patient. In addition, administration of the effective doseis such that the concentration of the compound within the plasma of thepatient normally does not exceed 500 ng/mL and frequently does notexceed 100 ng/mL.

IV. Methods of Using the Compounds and/or Pharmaceutical Compositions

The compounds can be used to treat cancers, including blood-bornecancers and solid tumors. Representative cancers include drug-resistantovarian cancer, drug resistant T-cell lymphoma, leukemia, non-small celllung, colon, central nervous system (CNS), melanoma, renal, ovarian,breast and prostate cancer.

The compounds can also be used as adjunct therapy in combination withexisting therapies in the management of the aforementioned types ofcancers. In such situations, it is preferably to administer the activeingredients to in a manner that optimizes effects upon cancer cells,including drug resistant cancer cells, while minimizing effects uponnormal cell types. While this is primarily accomplished by virtue of thebehavior of the compounds themselves, this can also be accomplished bytargeted drug delivery and/or by adjusting the dosage such that adesired effect is obtained without meeting the threshold dosage requiredto achieve significant side effects.

The following examples are provided to illustrate the present invention,and should not be construed as limiting thereof. In these examples, allparts and percentages are by weight, unless otherwise noted. Reactionyields are reported in mole percentages.

EXAMPLES

The following examples are provided to illustrate the present inventionand should not be construed as limiting the scope thereof.

Example 1 Tubulin Binding Assay (Measurement of Dissociation Constant(Kd) of Noscapine and 9-Aminonoscapine with Tubulin)

Noscapine and 9-aminonoscapine (0-150 μM) were incubated with 2 μMtubulin in 25 mM PIPES, pH 6.8, 3 mM MgSO₄, and 1 mM EGTA for 30 min at25° C. The fluorescence intensities of tubulin in the absence andpresence of different concentrations of the agents were monitored in aJASCO FP-6500 spectrofluorometer (JASCO, Tokyo, Japan) using a cuvetteof 0.3-cm path length.

The excitation wavelength was 295 nm. The inner filter effects werecorrected using a formula:

Fcorrected−Fobserved=antilog[(Aex−Aem)/2],

where Aex and Aem are the absorbance at the excitation and emissionwavelengths.

The dissociation constant (Kd) was estimated using the followingequation.

ΔF=ΔFmaxL/(Kd+L)

where, ΔF is change in the fluorescence intensity of the tubulin uponbinding to noscapine or 9-aminonoscapine, ΔFmax is the maximum change inthe fluorescence intensity of the protein when it is fully bound withnoscapine or 9-aminonoscapine, and L is the concentration of the ligand.

The ΔFmax value was calculated by the GraphPad Prism 5 software. ΔF wascalculated by subtracting the fluorescence intensity of tubulin in theabsence of noscapine or 9-aminonoscapine from the fluorescence intensityof tubulin in the presence of different concentrations of noscapine or9-aminonoscapine.

The data were statistically analyzed and curve fitted using GraphPadPrism 5 software.

Sedimentation Assay

Tubulin solution was centrifuged for 10 min at 80000 g at 4° C. Solubletubulin was measured by Bradford method. Tubulin (10 μM) was mixed withdifferent concentrations of 9-amino-Noscapine (0, 50, 100, 15 μM) in theassembly buffer (100 mM PIPES at pH 6.8, 3 mM MgSO₄, 1 mM EGTA, 1 mMGTP, and 1 M sodium glutamate). A reaction control with DMSO was alsoset up.

Polymerization was carried on by maintaining the temperature at 37° C.in the water bath for 30 min. After polymerization, the reaction mixturewas centrifuged at 120000 g at 30° C. for 30 min. The soluble tubulincontent was measured by Bradford method. Polymer mass of tubulin wasfound out by deducting soluble tubulin mass from the total tubulincontent.

Results

9-Aminonoscapine has Higher Tubulin Binding Activity than Noscapine

Tubulin, like many other proteins, contains fluorescent amino acids liketryptophans and tyrosines and the intensity of the fluorescence emissionis dependent upon the micro-environment around these amino acids in thefolded protein. Agents that bind tubulin typically change themicro-environment and the fluorescent properties of the target protein.Measuring these fluorescent changes has become a standard method fordetermining the binding properties of tubulin ligands including theclassical compound colchicine. This standard method was employed todetermine the dissociation constant (Kd) between tubulin and noscapineor 9-aminonoscapine.

It was determined that noscapine and 9-aminonoscapine both reduced theintrinsic fluorescence of tubulin in a concentration-dependent manner(FIGS. 1A and B). The double reciprocal plots yielded a dissociationconstant (Kd) of 152±1 μM for noscapine binding to tubulin (FIG. 1C).9-aminonoscapine was found to bind to tubulin with a Kd of 14±1 μM (FIG.1D) suggesting that 9-aminonoscapine has a significantly higher bindingaffinity for tubulin than that of noscapine.

9-Aminoscapine has Negligible Effect on Tubulin Polymerization

Tubulin was polymerized in presence or absence of 9-aminonoscapine toobserve its effect on polymer formation. Polymer mass of tubulin wasmeasured by sedimentation assay. Before polymerization soluble tubulincomprises total tubulin. Total tubulin content was measured by measuringsoluble tubulin by Bradford method. After polymerization, solubletubulin content was measured and deducted from total tubulin content tofind out the polymeric tubulin level.

TABLE 1 Concentration of 9-aminonoscapine Average % increase in polymerlevel 25 μM  2.8 ± 0.98 50 μM 3.2 ± 3.6 100 μM  6.3 ± 3.8

Table 1 indicates that 9-aminonoscapine had negligible effect on theassembly of tubulin into microtubules in vitro. 100 μM 9-amino-noscapinecaused only 6.3±3.8% increase in polymeric tubulin level, indicatingthat it induced aggregation of tubulin at very high concentrations

Having hereby disclosed the subject matter of the present invention, itshould be apparent that many modifications, substitutions, andvariations of the present invention are possible in light thereof. It isto be understood that the present invention can be practiced other thanas specifically described. Such modifications, substitutions andvariations are intended to be within the scope of the presentapplication.

NCI 60-tumor cell line data: 9-aminonocapine is an effective anti-canceragent that blocks cellular proliferation of a wide variety of cancercells.

9-aminonoscapine is a potent anticancer agents that inhibits theproliferation of various human cancer cells. The panel of 60 human tumorcell lines is organized into subpanels representing leukemia, non-smallcell lung, colon, CNS, melanoma, renal, ovarian, breast and prostratecancer lines. Cells were treated with 9-aminonoscapine at increasinggradient concentrations for 48 h. The IC₅₀ values, which stand for thedrug concentration needed to prevent cell proliferation by 50% was thenmeasured using an in vitro Sulforhodamine B assay. Panels showbar-graphically the comparison of IC₅₀ values of 9-aminonoscapine (blackbars) for cancer cell lines of various tissue origins.

Having hereby disclosed the subject matter of the present invention, itshould be apparent that many modifications, substitutions, andvariations of the present invention are possible in light thereof. It isto be understood that the present invention can be practiced other thanas specifically described. Such modifications, substitutions andvariations are intended to be within the scope of the presentapplication.

1-2. (canceled)
 3. A method of treating or preventing cancer in a patient, comprising administering an effective amount of a compound having the following formula:

and pharmaceutically acceptable salts and prodrugs thereof to a patient in need of treatment thereof.
 4. The method of claim 3, wherein the cancer is selected from the group consisting of drug-resistant ovarian cancer, drug resistant T-cell lymphoma, leukemia, non-small cell lung, colon, central nervous system (CNS), melanoma, renal, ovarian, breast and prostate cancer.
 5. The method of claim 4, wherein the cancer is drug-resistant ovarian cancer or T-cell lymphoma.
 6. The method of claim 3, wherein the cancer is a drug-resistant cancer. 7-9. (canceled)
 10. The method of claim 3, wherein the prodrug is

11-15. (canceled) 